1. Field of the Invention
The present invention provides crystal structures of cellular molecules that play important roles in immunity, phosphorylation events, and disease initiation mechanisms. The isolated crystals and methods for crystallization thereof, are also important in identifying small molecule interactions with cellular molecules for new drug discovery.
2. Background
Protein tyrosine phosphorylation is an important molecular switching mechanism that regulates a variety of cellular functions including cell proliferation, differentiation, and activation. Tyrosine phosphorylation is not only an essential part of the signal transduction mediated by various growth factor receptors, but it is also involved in intracellular signal transduction and nuclear cell cycle regulation. Disturbances of these processes are known to be causes of cancer. For example, overexpression and/or hyper-activation of many protein tyrosine kinases are oncogenic. Thus, knowledge about the regulation of protein tyrosine phosphorylation provides valuable information about the control of basic cellular processes and is essential to understanding the generation of cancer. In this regard, the ultimate goal of many converging lines of research is the eventual development of rational therapeutic agents.
Protein tyrosine phosphorylation is a reversible process that involves both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Protein phosphorylation is now well recognized as an important mechanism utilized by cells to transduce signals during different stages of cellular function (Fischer et al, Science 253:401–6 (1991); Flint et al., The EMBO J. 12:1937–46 (1993)). There are at least two major classes of phosphatases: (1) those that dephosphorylate proteins (or peptides) that contain a phosphate group(s) on a serine or threonine moiety (termed Ser/Thr phosphatases) and (2) those that remove a phosphate group(s) from the amino acid tyrosine (termed protein tyrosine phosphatases or PTPases). The PTPases can be further subdivided into two groups: a) intracellular or nontransmembrane PTPases and b) receptor-type or transmembrane PTPases.
Most known intracellular type PTPases contain a single conserved catalytic phosphatase domain consisting of 220–240 amino acid residues. The regions outside the PTPase domains are believed to play important roles in localizing the intracellular PTPases subcellularly (Mauro, L. J. and Dixon, J. E. TIBS 19: 151–155 (1994)). The first intracellular PTPase to be purified and characterized was PTP1B which was isolated from human placenta (Tonks et al., J. Biol. Chem. 263: 6722–6730 (1988)). Other examples of intracellular PTPases include (1) T-cell PTPase (Cool et al. Proc. Natl. Acad. Sci. USA 86: 5257–5261 (1989)), (2) rat brain PTPase (Guan et al., Proc. Natl. Acad. Sci. USA 87:1501–1502 (1990)), (3) neuronal phosphatase STEP (Lombroso et al., Proc. Natl. Acad. Sci. USA 88: 7242–7246 (1991)), (4) ezrin-domain containing PTPases: PTPMEG1 (Guet al., Proc. Natl. Acad. Sci. USA 88: 5867–57871 (1991)), PTPH1 (Yang and Tonks, Proc. Natl. Acad. Sci. USA 88: 5949–5953 (1991)), PTPD1 and PTPD2 (M. Oller et al., Proc. Natl. Acad. Sci. USA 91: 7477–7481 (1994)), FAP-1/BAS (Sato et al., Science 268: 411–415 (1995); Banville et al., J. Biol. Chem. 269: 22320–22327 (1994); Maekawa et al., FEBS Letters 337: 200–206 (1994)), and SH2 domain containing PTPases: PTP1C/SH-PTP1/SHP-1 (Plutzky et al., Proc. Natl. Acad. Sci. USA 89: 1123–1127 (1992); Shen et al., Nature Lond. 352: 736–739 (1991)) and PTP1D/Syp/SH-PTP2/SHP-2 (Vogel et al., Science 259: 1611–1614 (1993); Feng et al., Science 259: 1607–1611 (1993); Bastein et al., Biochem. Biophys. Res. Comm. 196: 124–133 (1993)).
Receptor-type PTPases (RPTPases) consist of a) a putative ligand-binding extracellular domain, b) a transmembrane segment, and c) an intracellular catalytic region. The structures and sizes of the putative ligand-binding extracellular domains of receptor-type PTPases are quite divergent. In contrast, the intracellular catalytic regions of receptor-type PTPases are very homologous to each other and to the intracellular PTPases. Most receptor-type PTPases have two tandemly duplicated catalytic PTPase domains.
The first receptor-type PTPases to be identified were (1) CD45 also known as Leukocyte Common Antigen (LCA) ((Ralph, S. J., EMBO J. 6: 1251–1257 (1987)) and (2) Leukocyte common Antigen Related (LAR)(Streuli et al., J. Exp. Med. 168: 1523–1530 (1988)) that were recognized to belong to this class of enzymes based on homology to PTP1B (Charbonneau et al., Proc. Natl. Acad. Sci. USA 86: 5252–5256 (1989)). CD45 is a member of a family of high molecular weight glycoproteins, is one of the most abundant leukocyte cell surface glycoproteins, and appears to be exclusively expressed upon cells of the hematopoietic system (Trowbridge and Thomas, Ann. Rev. Immunol. 12: 85–116 (1994)).
The identification of CD45 and LAR as members of the PTPase family was quickly followed by identification and cloning of several different members of the receptor-type PTPase group. Thus, 5 different PTPases, (3) PTPα, (4) PTPβ, (5) PTPδ, (6) PTPε, and (7) PTPζ, were identified in one early study (Krueger et al., EMBO J. 9: 3241–3252 (1990)). Other examples of receptor-type PTPases include (8) PTPγ (Barnea et al., Mol. Cell. Biol. 13: 1497–1506 (1995)) which, like PTPζ (Krueger and Saito, Proc. Natl. Acad. Sci. USA 89: 7417–7421 (1992)) contains a carbonic anhydrase-like domain in the extracellular region, (9) PTPμ (Gebbink et al., FEBS Letters 290: 123–130 (1991)), (10) PTPκ (Jiang et al., Mol. Cell. Biol. 13: 2942–2951 (1993)). Based on structural differences the receptor-type PTPases may be classified into subtypes (Fischer et al., Science 253: 401–406 (1991)): (I) CD45; (II) LAR, PTPδ, (11) PTPσ; (III) PTPβ, (12) SAP-1 (Matozaki et al., J. Biol. Chem. 269: 2075–2081 (1994)), (13) PTP-U2/GLEPP1 (Seimiya et al., Oncogene 10: 1731–1738 (1995); Thomas et al., J. Biol. Chem. 269: 19953–19962 (1994)), and (14) DEP-1; (IV) PTPα, PTPε. All receptor-type PTPases except Type IV contain two PTPase domains. Novel PTPases are continuously identified, and it is anticipated that more than 500 different species will be found in the human genome, i.e. close to the predicted size of the protein tyrosine kinase superfamily (Hanks and Hunter, FASEB J. 9: 576–596 (1995)).
Considerable information regarding the interactions of specific PTKases in various cellular pathways has led to a general understanding of their roles and the regulation of such. However, much less is known about the specific functions or the control of PTPases in these pathways.
There is thus a need to determine the structural basis of the function of significant representatives of protein tyrosine phosphatases such as the human leukocyte PTPases, LAR and CD45 (CD45).